Biochemistry Lecture 8 Enzyme Kinetics Why Enzymes Higher

Biochemistry Lecture 8 Enzyme Kinetics

Why Enzymes? • • Higher reaction rates Greater reaction specificity Milder reaction conditions Capacity for regulation • Metabolites have many potential pathways of decomposition • Enzymes make the desired one most favorable

Enzymatic Substrate Selectivity No binding Binding but no reaction Example: Phenylalanine hydroxylase

Enzymes Affect Reaction Rates • Catalyst • Protein (globular) or RNA • Classified based on the reaction catalyzed 4

Potential Energy H 2 O + CO 2 HOCO 2– Ea Ea' Reaction + H+

How to Lower G ? Enzymes bind transition states best

How Do Enzymes Stabilize the Transition State and Increase Reaction Rate? Conserved active site amino acid residues in the enzyme can help with orientation of the substrate and stabilizing transition state 9

erine Protease Mechanis 10

How Do Enzymes Stabilize the Transition State and Increase Reaction Rate? Conserved active site amino acid residues, metal ions and organic molecules in the enzyme can help with orientation of the substrate and stabilizing transition 11 state

How is TS Stabilization Achieved? – acid-base catalysis: give and take protons – covalent catalysis: change reaction paths – metal ion catalysis: use redox cofactors, p. Ka shifters – electrostatic catalysis: preferential interactions with TS End result? Rate enhancements of 105 to 1017!

How is TS Stabilization Achieved? – covalent catalysis: change reaction paths

How to Lower Enzymes organizes reactive groups into proximity G ?

Enzyme Kinetics • Kinetics is the study of the rate at which compounds react • Rate of enzymatic reaction is affected by – Enzyme – Substrate – Effectors – Temperature

How to Do Kinetic Measurements

Steady-State Assumption 42

What equation models this behavior? Michaelis-Menten Equation

Michaelis-Menten Kinetics k 1 k 2 E + S E + P k-1 Derived using a few assumptions: – steady state assumption: formation of ES = breakdown of ES (until a significant amount of S has been consumed). – consider initial velocity at early time-points, [P] = 0: rate of reaction depends exclusively on the breakdown of ES (k-2 can be ignored). – free ligand assumption: [S] is in such excess that its decrease in concentration when forming 46 ES is negligible (total [S] = free [S] + [ES]).

Simple Enzyme Kinetics • The final form in case of a single substrate is • kcat (turnover number): how many substrate molecules can one enzyme molecule convert per second • Km (Michaelis constant): an approximate measure of substrate’s affinity for enzyme • Microscopic meaning of Km and kcat depends on the details of the mechanism

Calculating Vmax and Km: The Double Reciprocal Plot 49

Enzyme Inhibition Inhibitors are compounds that decrease enzyme’s activity • Irreversible inhibitors (inactivators) react with the enzyme - one inhibitor molecule can permanently shut off one enzyme molecule - they are often powerful toxins but also may be used as drugs • Reversible inhibitors bind to, and can dissociate from the enzyme - they are often structural analogs of substrates or products - they are often used as drugs to slow down a specific enzyme • Reversible inhibitor can bind: – To the free enzyme and prevent the binding of the substrate – To the enzyme-substrate complex and prevent the reaction

Reversible Inhibition Competitive • No change in Vmax; apparent increase in Km • Lineweaver-Burk: lines intersect at the y-axis 52

Reversible Inhibition Uncompetitive • Decrease in Vmax; apparent decrease in Km • No change in Km/Vmax • Lineweaver-Burk: lines are parallel 54

Reversible Inhibition – Mixed Inhibition • Decrease in Vmax; apparent change in Km • Lineweaver-Burk: lines intersect left from the yaxis • Noncompetitive inhibitors are mixed inhibitors such that there is no change in Km 56

Acetylcholinesterase

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